Pharmaceutical composition for the prevention and treatment of cardiovascular disease comprising the peptide having the ability to inhibit angiotensin-1 converting enzyme as an active ingredient

ABSTRACT

The present invention relates to a peptide separated from the fraction of oyster enzyme hydrate displaying the ability of suppressing angiotensin converting enzyme (ACE) and a pharmaceutical composition for the prevention and treatment of cardiovascular disease comprising the said peptide as an active ingredient. Particularly, the peptide separated from the fraction of the oyster enzyme hydrate of the present invention significantly inhibits ACE activity, and thus brings blood pressure regulating effect and antihypertensive effect. Therefore, the fraction of the oyster enzyme hydrate of the invention or the peptide separated from the same can be effectively used as an active ingredient of a pharmaceutical composition for the prevention or treatment of cardiovascular disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT/KR2013/007598 filedAug. 23, 2013 which claims the benefit of Korean patent applicationsKR-10-2012-0092738 filed Aug. 24, 2012 and KR-10-2013-0100232 filed Aug.23, 2013, the contents of each of which are incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a peptide separated from the fractionof the oyster enzyme hydrate displaying the ability of suppressingangiotensin converting enzyme (ACE) and a pharmaceutical composition forthe prevention and treatment of cardiovascular disease comprising thesaid peptide as an active ingredient.

2. Description of the Related Art

According to the rapid economic development, the pattern and the aspectof disease have also been changed, so that cardiovascular disease isremarkably increasing, for example arteriosclerosis, hypertension,angina pectoris, myocardial infarction, ischemic heart disease, heartfailure, complications caused by transluminal coronary angioplasty,cerebral infarction, cerebral hemorrhage, and stroke are in increase.According to the ranking of cause of death reported by Statistics Koreain 2010, cardiovascular disease is ranked as the second topmost cause ofdeath in Korea, which is next to malignant tumor. Particularly, deathrate of cardiovascular disease is significantly increased in male over55 years of age and in female over 65 years of age.

The most representative cardiovascular disease, hypertension, takes15˜20% of total adult disease, making it a world-wide health issue. Eventhough there is no specific symptoms in hypertension patients, it ishighly required to control and treat the disease because the risk of thesame complications as shown in other cardiovascular diseases such asarteriosclerosis, stroke, myocardial infarction and end-stage renaldisease is still high. Hypertension is a chronic disease that requireslife-long treatment, suggesting that social and economical loss isgreat.

The cause of hypertension is still unknown, but is presumed to bedeveloped by genetic factors such as family history, races, salt intake,insulin resistance, and obesity and/or environmental factors such asexcessive drinking and aging, etc. Among many factors that are involvedin raising blood pressure, renin-angiotensin-aldosterone cycle has beenknown to play an important role in regulating blood pressure and bodyfluid level in vivo (Weiss, D. et. al (2001) Circulation 103: 448-454).

In particular, renin-angiotensin system (RAS) has been known to play animportant role in the development of essential hypertension which takes80% of total hypertension cases. In general, renin-angiotensin system isactivated when the blood flow rate or sodium level is dropped in thekidney or when the activity of sympathetic nervous system is increased.Renin secreted in juxtaglomerular cells in renal artery decomposesangiotensin into angiotensin I and then angiotensin I is converted intoangiotensin II inducing the contraction of blood vessel by angiotensinconverting enzyme (ACE). As a result, angiotensin II regulates bloodpressure by increasing aldosterone synthesis and by neuroregulation. ACEalso decomposes bradykinin having vasodilating activity so as toinactivate bradykinin.

Therefore, angiotensin converting enzyme inhibitor (ACE inhibitor) isexpected to be able to treat or prevent cardiovascular diseases such ashypertension, heart disease, arteriosclerosis, or cerebral hemorrhage,considering that ACE has a great effect on blood pressure increase, onwhich therefore studies have been focused. Particularly, variousclinical attempts have been made to confirm if the decrease of the casesand even the death rate of chronic kidney disease, arteriosclerosis, andheart attack could be achieved by such ACE inhibitor and the resultsthereby have been reported.

Based on the above reports, chemically synthesized angiotensinconverting enzyme inhibitors such as ramipril, captopril, enarapil,risinopril, fosinoril, and spirapril are commercialized and used ashypertension treating agents. However, these compounds are so easilydecomposed in a pharmaceutical administration form, that means stabilityis low, and they cause side effects by affecting other cells includingweakness of whole body, vomiting, cough, headache, anorexia, and tastedisorder, etc. (Lim S D. et. al (2008) Korean J. Food Sci. Ani. Resour,28(5): 587-595). Thus, it is required to develop a natural ACE inhibitorthat has increased stability but has less side effects in vivo.

Oyster, nicknamed “the milk from the ocean,” has been known as anexcellent health food. Even in Western countries, where live sea food isnot eaten as often as in Asian countries, live oyster is eaten. Asnutrition factors, oyster contains glycogen, taurine, protein, vitamin,and various minerals, making an excellent substance for health food.Oyster is also effective in strengthening the functions of heart andliver, in treating hypertension and arteriosclerosis, and in preventingheart disease. Since oyster contains a plenty of selenium, it not onlyhas detoxication activity for heavy metals but also is known as thetonic and stamina food increasing the functions of heart, liver,pancreas, and other organs.

As for the known natural ACE inhibitors so far, Korean PatentPublication No. 10-2012-0092735 describes the composition for inhibitingangiotensin converting enzyme, or having antihypertensive orantidiabetic property, comprising Capsosiphon fulvescens extract treatedby enzyme. Korean Patent No. 10-1275766 describes the composition forinhibiting angiotensin converting enzyme, or having antihypertensive orantiobese property, comprising protein extract of abalone intestine

The peptides having ACE inhibiting activity have been identified fromthe enzyme hydrolysates of natural substances. Among them,valine-tyrosine, the peptide originated from sardine protein has beenconfirmed to have the activity of reducing blood pressure in mildhypertension patients (Shimizu, M (1994) Melbourne Sept, 18), because ofwhich it has been approved as the first individual case-authorizedhealth food in Korea. Korean Patent No. 10-1106303 describes the ACEinhibiting activity of the peptide prepared from oyster. However, theneed for the development of natural substance derived functionalpeptides is still in increase.

Therefore, the present inventors tried to identify a peptide having ACEinhibiting activity from the enzyme hydrolysates of natural substances.As a result, the present inventors obtained a novel peptide having ACEinhibiting activity by extracting, separating, and purifying the peptidefrom oyster enzyme hydrate. Thereafter, the present inventors completedthis invention by confirming that the novel peptide identified by theinventors had the effect of regulating blood pressure and of preventinghypertension thereby so that the peptide separated from oyster enzymehydrate could be effectively used as an active ingredient of apharmaceutical composition for the prevention or treatment ofcardiovascular disease.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a peptide havingangiotensin converting enzyme (ACE) inhibiting activity comprising oneof the amino acid sequences represented by SEQ. ID. NO: 1˜NO: 12.

It is another object of the present invention to provide apharmaceutical composition for the prevention and treatment ofcardiovascular disease comprising the fraction of the oyster enzymehydrate containing the said peptide as an active ingredient.

It is also an object of the present invention to provide apharmaceutical composition for the prevention or treatment ofcardiovascular disease comprising at least one of the said peptides asan active ingredient.

It is further an object of the present invention to provide a healthfood for the prevention or improvement of cardiovascular diseasecomprising the fraction of the oyster enzyme hydrate containing the saidpeptide as an active ingredient.

It is also an object of the present invention to provide a health foodfor the prevention or improvement of cardiovascular disease comprisingat least one of the said peptides as an active ingredient.

It is also an object of the present invention to provide a method fortreating cardiovascular disease containing the step of administering thefraction of the oyster enzyme hydrate comprising the said peptide to asubject having cardiovascular disease.

It is also an object of the present invention to provide a method fortreating cardiovascular disease containing the step of administering atleast one of the said peptides to a subject having cardiovasculardisease.

It is also an object of the present invention to provide a method forpreventing cardiovascular disease containing the step of administeringthe fraction of the oyster enzyme hydrate comprising the said peptide toa subject.

It is also an object of the present invention to provide a method forpreventing cardiovascular disease containing the step of administeringat least one of the said peptides to a subject.

It is also an object of the present invention to provide a use of thefraction of the oyster enzyme hydrate comprising the said peptide as apharmaceutical composition for the prevention or treatment ofcardiovascular disease.

It is also an object of the present invention to provide a use of atleast one of the said peptides as a pharmaceutical composition for theprevention or treatment of cardiovascular disease.

It is also an object of the present invention to provide a use of thefraction of the oyster enzyme hydrate comprising the said peptide as ahealth food for the prevention or improvement of cardiovascular disease.

It is also an object of the present invention to provide a use of atleast one of the said peptides as a health food for the prevention orimprovement of cardiovascular disease.

To achieve the above objects, the present invention provides a peptidehaving angiotensin converting enzyme (ACE) inhibiting activitycomprising one of the amino acid sequences represented by SEQ. ID. NO:1˜NO: 12.

The present invention also provides a pharmaceutical composition for theprevention and treatment of cardiovascular disease comprising thefraction of the oyster enzyme hydrate containing the said peptide as anactive ingredient.

The present invention further provides a pharmaceutical composition forthe prevention or treatment of cardiovascular disease comprising atleast one of the said peptides as an active ingredient.

The present invention also provides a health food for the prevention orimprovement of cardiovascular disease comprising the fraction of theoyster enzyme hydrate containing the said peptide as an activeingredient.

The present invention also provides a health food for the prevention orimprovement of cardiovascular disease comprising at least one of thesaid peptides as an active ingredient.

The present invention also provides a method for treating cardiovasculardisease containing the step of administering the fraction of the oysterenzyme hydrate comprising the said peptide to a subject havingcardiovascular disease.

The present invention also provides a method for treating cardiovasculardisease containing the step of administering at least one of the saidpeptides to a subject having cardiovascular disease.

The present invention also provide a method for preventingcardiovascular disease containing the step of administering the fractionof the oyster enzyme hydrate comprising the said peptide to a subject.

The present invention also provides a method for preventingcardiovascular disease containing the step of administering at least oneof the said peptides.

The present invention also provides a use of the fraction of the oysterenzyme hydrate containing the said peptide as a pharmaceuticalcomposition for the prevention or treatment of cardiovascular disease.

The present invention also provides a use of at least one of the saidpeptides as a pharmaceutical composition for the prevention or treatmentof cardiovascular disease.

The present invention also provides a use of the fraction of the oysterenzyme hydrate containing the said peptide as a health food for theprevention or improvement of cardiovascular disease.

In addition, the present invention provides a use of at least one of thesaid peptides as a health food for the prevention or improvement ofcardiovascular disease.

ADVANTAGEOUS EFFECT

The peptide of the present invention that shows angiotensin convertingenzyme (ACE) inhibiting activity and has been separated from thefraction of the oyster enzyme hydrate has the effect of regulating bloodpressure and of preventing hypertension thereby, so that the fraction ofthe oyster enzyme hydrate or the peptide isolated therefrom can beeffectively used as an active ingredient for a pharmaceuticalcomposition for the prevention or treatment of cardiovascular disease.

BRIEF DESCRIPTION OF THE DRAWINGS

The application of the preferred embodiments of the present invention isbest understood with reference to the accompanying drawings, wherein:

FIG. 1 is a set of graphs illustrating the purification of the oysterenzyme hydrate. A indicates the purification by anion exchangechromatography, B indicates the purification by size exclusionchromatography of the fraction 4 obtained from the above anion exchangechromatography, and C indicates the purification by reverse-phasechromatography.

FIG. 2 is a set of graphs illustrating the mass and the amino acidsequence of the peptide separated from the oyster enzyme hydrate.

FIG. 3 is a graph illustrating the effect of in vivo blood pressureregulation by single administration of the oyster enzyme hydrate.

FIG. 4 is a graph illustrating the effect of in vivo blood pressureregulation by single administration of the functional peptide.

FIG. 5 is a graph illustrating the effect of in vivo blood pressureregulation by repeated administration of the oyster enzyme hydrate andthe functional peptide.

FIG. 6 is a graph illustrating the inhibitory effect of the oysterenzyme hydrate and the functional peptide on the level of bloodangiotensin converting enzyme (ACE).

FIG. 7 is a graph illustrating the inhibitory effect of the oysterenzyme hydrate and the functional peptide on the level of bloodangiotensin-II.

FIG. 8 is a graph illustrating the preventive effect of the oysterenzyme hydrate and the functional peptide on hypertension in vivo.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, the present invention is described in detail.

The present invention provides a peptide having angiotensin convertingenzyme (ACE) inhibiting activity comprising one of the amino acidsequences represented by SEQ. ID. NO: 1˜NO: 12.

The peptide is preferably separated from the oyster enzyme hydrate, butnot always limited thereto and the peptide can be originated from othernatural sources or can be synthesized.

For the separation of the peptide, any conventional method such asultrafiltration and chromatography which are well known to those in theart is preferred and chromatography is more preferred. Particularly,anion exchange chromatography, size exclusion chromatography,reverse-phase chromatography or high performance liquid chromatography(HPLC) is preferably used, but not always limited thereto.

For the synthesis of the peptide, the conventional peptide synthesismethod such as chemical synthesis method is preferred (W. H. Freeman andCo., Proteins; structures and molecular principles, 1983). Particularly,solution phase peptide synthesis, solid-phase peptide syntheses,fragment condensation, F-moc, or T-BOC chemical method is morepreferred, and among these methods, solid-phase peptide synthesis ismost preferred, but not always limited thereto.

The enzyme herein is preferably the conventional protease that iswell-informed to those in the art, and more preferably protamex orneutrase, but not always limited thereto. The addition of the enzyme isperformed stepwise. Precisely, protamex is first reacted to induceinactivation and then neutrase is added, but not always limited theretoand protamex and neutrase can be added simultaneously. The preferableconcentration of the enzyme is 0.1˜10% by the concentration of theoyster enzyme hydrate protein. The inactivation of the enzyme ispreferably induced at 20˜100° C. for 10˜120 minutes, but not alwayslimited thereto and the inactivation condition can be modified by thekind of enzyme.

In a preferred embodiment of the present invention, the presentinventors hydrolyzed the oyster protein by using a protease in order toprepare the oyster enzyme hydrate. Then, chromatography was performed topurify the functional peptide having ACE inhibiting activity from theoyster enzyme hydrate. As a result, 7 samples displaying ACE inhibitingactivity were selected (see FIG. 1, Tables 1 and 2).

To screen the functional peptide having ACE inhibiting activity from thesamples, mass and amino acid sequence of the peptide were confirmed. Asa result, the peptide comprising one of the amino acid sequencesrepresented by SEQ. ID. NO: 1˜NO: 12 was selected (see FIG. 2 and Table3). The peptide was chemically synthesized and then ACE inhibitingactivity and cytotoxicity were investigated. As a result, all thepeptides of the invention demonstrated ACE inhibiting activity withoutcytotoxicity (see Table 4).

Therefore, the peptide of the present invention can be effectively usedas an ACE activity inhibitor since it demonstrates a significant ACEinhibiting activity without cytotoxicity.

The peptide of the present invention can be prepared by the belowgenetic engineering method. First, DNA sequence encoding the saidpeptide was constructed according to the conventional method. DNAsequence can be constructed by PCR using proper primers. DNA sequencecan also be synthesized by other standard methods informed to those inthe art such as the method using an automatic DNA synthesizer (forexample, the product of Biosearch or Applied Biosystems). The DNAsequence was inserted in the vector containing one or more expressionregulating sequences (for example, promoter, enhancer, etc.) that wereoperably linked to the said DNA sequence to express thereof, resultingin the construction of the recombinant expression vector. Host cellswere transformed with the constructed recombinant expression vector andthe generated transformant was cultured in a proper medium under thesuggested condition to induce the expression of the DNA sequence. Thepure peptide encoded by the DNA sequence was collected by theconventional method well known to those in the art (for example,chromatography). The “pure peptide” indicates the peptide that does notcontain any other proteins originated from the host. The geneticengineering method used for synthesis of the peptide herein referred to:Maniatis et al., Molecular Cloning; A laboratory Manual, Cold SpringHarbor laboratory, 1982; Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, N.Y., Second (1998) andThird (2000) Edition; Gene Expression Technology, Method in Enzymology,Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink(eds.), Academic Press, San Diego, Calif., 1991; and Hitzeman et al., J.Biol. Chem., 255:12073-12080, 1990.

The peptide of the present invention can be administered parenterallyand be used in general forms of pharmaceutical formulation. Theparenteral administration herein includes intrarectal, intravenous,intraperitoneal, intramuscular, intra-arterial, percutaneous,intranasal, inhalation, ocular, and subcutaneous administration.

That is, the peptide of the present invention can be prepared forparenteral administration by mixing with generally used diluents orexcipients such as fillers, extenders, binders, wetting agents,disintegrating agents and surfactants. Formulations for parenteraladministration are sterilized aqueous solutions, water-insolubleexcipients, suspensions, emulsions, lyophilized preparations andsuppositories. Water insoluble excipients and suspensions can contain,in addition to the active compound or compounds, propylene glycol,polyethylene glycol, vegetable oil like olive oil, injectable ester likeethylolate, etc. Suppositories can contain, in addition to the activecompound or compounds, witepsol, macrogol, tween 61, cacao butter,laurin butter, glycerogelatin, etc.

The peptide of the present invention can be mixed with manypharmaceutically acceptable carriers such as physiological saline ororganic solvent, and can additionally include carbohydrates such asglucose, sucrose or dextran, antioxidants such as ascorbic acid orglutathion, chelating agents, low molecular proteins or otherstabilizers to enhance stability or absorptiveness.

The effective dosage of the peptide of the present invention is 0.01˜100mg/kg per day, and preferably 0.1˜10 mg/kg per day, and administrationfrequency is preferably 1˜3 times a day.

The effective dose of the peptide of the present invention can beadministered in the form of bolus, by single dose having relativelyshort period of infusion or by multiple dose of fractionated treatmentprotocol for a long term. The decision of an effective dosage of thepeptide depends on the administration pathway, treatment times, age andother conditions of a patient, etc. Therefore, any expert who hasknowledge on this field can decide the effective dosage of the peptideof the present invention.

The present invention also provides a pharmaceutical composition for theprevention and treatment of cardiovascular disease comprising thefraction of the oyster enzyme hydrate containing the peptide comprisingone of the amino acid sequences represented by SEQ. ID. NO: 1˜NO: 12 asan active ingredient.

The fraction herein is preferably up to 10 kD, but not always limitedthereto.

The said cardiovascular disease is preferably one or more diseasesselected from the group consisting of hypertension, heart disease,stroke, thrombosis, angina pectoris, heart failure, myocardialinfarction, atherosclerosis, and arteriosclerosis, but not alwayslimited thereto.

In another preferred embodiment of the present invention, the inventorsadministered the peptide to the hypertension mouse model via single ormultiple administrations in order to investigate the blood pressureregulating activity of the peptide of the invention. As a result, theoyster enzyme hydrate and YA peptide were confirmed to have thesignificant antihypertensive activity (see FIGS. 4 and 5), andaccordingly the levels of blood ACE and angiotensin-II weresignificantly decreased (see FIGS. 6 and 7).

To investigate the antihypertensive effect of the peptide of theinvention in vivo, the present inventors administered the peptide of theinvention to the mouse induced with hypertension, followed byexamination of the antihypertensive effect. As a result, blood pressurein the mouse was significantly lowered (see FIG. 8).

Therefore, the fraction of the oyster enzyme hydrate containing thepeptide comprising one of the amino acid sequence represented by SEQ.ID. NO: 1˜NO: 12 had the blood pressure regulating activity and therebyantihypertensive effect, so that the fraction can be effectively used asan active ingredient of a pharmaceutical composition for the preventionor treatment of cardiovascular disease.

The composition of the present invention can be prepared for parenteraladministration by mixing with generally used diluents or excipients suchas fillers, extenders, binders, wetting agents, disintegrating agentsand surfactants.

In addition to the ingredients mentioned above, the pharmaceuticalcomposition of the present invention can include in a variety ofnutrients, vitamins, minerals, flavors, coloring agents, pectic acid andits salts, alginic acid and its salts, organic acid, protectivecolloidal viscosifiers, pH regulators, stabilizers, antiseptics,glycerin, alcohols, carbonators which used to be added to soda, etc. Thesaid carrier, excipient, or diluent can be selected from the groupconsisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol,erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calciumphosphate, calcium silcate, cellulose, methyl cellulose,microcrystalline cellulose, polyvinyl pyrrolidone, water,methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate,mineral oil, dextrine, calcium carbonate, propylene glycol, liquidparaffin, and saline, but not always limited thereto. All the mentionedingredients can be added singly or together.

The pharmaceutical composition of the present invention can further beprepared in suitable forms according to ingredients by following themethod represented in Remington's Pharmaceutical Science (the newestedition), Mack Publishing Company, Easton Pa.

The composition of the present invention is administered by thepharmaceutically effective dose. The term “pharmaceutically effectivedose” herein indicates the amount enough to treat the disease withapplicable, reasonable or risky concentration. The dose can bedetermined by considering many factors such as the type of disease,severity of the disease, activity of the drug, sensitivity to the drug,administration frequency and pathway, excretion, term of treatment,co-treatment drug and other factors regarded as relevant in themedicinal field. The composition of the present invention can beadministered as an individual drug or co-administered with other agentstogether, and at this time it can be administered serially orsimultaneously with the other agents. Single or multiple application isalso accepted. It is important to determine the dose to bring themaximum effect with the minimum amount without side effects, which canbe easily determined by those in the art.

The effective dose of the pharmaceutical composition of the presentinvention can be determined according to weight, age, gender, healthcondition, diet, administration frequency, administration method,excretion and severity of a disease. The preferable effective dose ofthe composition is 0.01˜1000 mg/kg per day, preferably 30˜500 mg/kg perday, and more preferably 50˜300 mg/kg per day, and administration timesare 1˜6 per day. However, the dose can be adjusted by consideringvarious factors such as administration pathway, severity of disease,patient's age, gender and weight, etc. Therefore, the preferable dosecannot limit the scope of the invention in any way.

The composition of the present can be administered alone or treatedtogether with surgical operation, radio-therapy, hormone therapy,chemo-therapy and biological regulators.

The present invention also provides a pharmaceutical composition for theprevention or treatment of cardiovascular disease comprising one or morepeptides composed of the amino acid sequences represented by SEQ. ID.NO: 1˜NO: 12 as an active ingredient.

The peptide of the present invention displays effective blood pressureregulating activity and antihypertensive effect thereby, so that any ofthose peptides of the invention can be effectively used as an activeingredient of a pharmaceutical composition for the prevention ortreatment of cardiovascular disease.

The present invention also provides a health food for the prevention orimprovement of cardiovascular disease comprising the fraction of theoyster enzyme hydrate containing the said peptide comprising one of theamino acid sequences represented by SEQ. ID. NO: 1 NO: 12 as an activeingredient.

The present invention also provides a health food for the prevention orimprovement of cardiovascular disease comprising at least one of thesaid peptides as an active ingredient.

The fraction of the oyster enzyme hydrate of the present invention orthe peptide isolated therefrom displays excellent ACE inhibitingactivity, suggesting that it could have efficient blood pressureregulating activity and antihypertensive effect thereby.

Therefore, the fraction of the oyster enzyme hydrate and the peptideseparated from the same can be effectively used as an active ingredientof a health food for the prevention or improvement of cardiovasculardisease.

The food herein is not limited. For example, the peptide of the presentinvention can be added to drinks, meats, sausages, breads, biscuits,rice cakes, chocolates, candies, snacks, cookies, pizza, ramyuns, gums,dairy products including ice cream, special nutrious food such as milkformulas or young children diet, processed meat products, fish products,Tofu, starch gel products, health supplement food, seasoning food suchas soy sauce, soybean paste, red pepper paste or mixed sauce, sauces,other processed food, pickles such as Kimchi or Jangajji, soups,beverages, alcohol drinks and vitamin complex, and in wide sense, almostevery food applicable in the production of health food can be included.

The “health food” in this invention indicates the value added food groupthat is designed to be functioning for a specific purpose or to induce afull expression of its composition via physical, biochemical, orbiotechnological technique, or the processed food designed to induce afull expression of each component of the food to regulate bio defensesystem or other regulation system involved in disease prevention andrecovery. More preferably, the health food of the invention indicatesthe food that can induce the full action of body regulating functionsinvolved in the prevention or improvement of cardiovascular disease. Thesaid health food can contain additives that can be accepted in the foodindustry and generally used carriers, excipients, and diluents in thepreparation of health food.

The fraction of the oyster enzyme hydrate of the present invention orthe peptide isolated therefrom can be used as food additive. In thatcase, the fraction of the oyster enzyme hydrate of the present inventionor the peptide isolated therefrom can be added as it is or as mixed withother food components according to the conventional method. The mixingratio of active ingredients can be regulated according to the purpose ofuse (prevention or health enhancement). In general, to produce healthfood or beverages, the fraction of the oyster enzyme hydrate of thepresent invention or the peptide isolated therefrom is added preferablyby 0.1˜90 weight part. However, if long term administration is requiredfor health and hygiene or regulating health condition, the content canbe lower than the above but higher content can be accepted as well sincethe fraction of the oyster enzyme hydrate of the present invention orthe peptide isolated therefrom has been proved to be very safe.

The composition for health beverages of the present invention canadditionally include various flavors or natural carbohydrates, etc, likeother beverages, in addition to the extract of crude drug complex. Thenatural carbohydrates above can be one of monosaccharides such asglucose and fructose, disaccharides such as maltose and sucrose,polysaccharides such as dextrin and cyclodextrin, and sugar alcoholssuch as xilytole, sorbitol and erythritol. Besides, natural sweeteningagents (thaumatin, stevia extract, for example rebaudioside A,glycyrrhizin, etc.) and synthetic sweetening agents (saccharin,aspartame, etc.) can be included as a sweetening agent. The content ofthe natural carbohydrate is preferably 1˜20 g and more preferably 5˜12 gin 100 ml of the composition.

In addition to the ingredients mentioned above, the fraction of theoyster enzyme hydrate of the present invention or the peptide isolatedtherefrom can include in a variety of nutrients, vitamins, minerals(electrolytes), flavors including natural flavors and synthetic flavors,coloring agents and extenders (cheese, chocolate, etc.), pectic acid andits salts, alginic acid and its salts, organic acid, protectivecolloidal viscosifiers, pH regulators, stabilizers, antiseptics,glycerin, alcohols, carbonators which used to be added to soda, etc. Thefraction of the oyster enzyme hydrate of the present invention or thepeptide isolated therefrom can also include natural fruit juice, fruitbeverages and/or fruit flesh addable to vegetable beverages. All thementioned ingredients can be added singly or together. The mixing ratioof those ingredients does not matter in fact, but in general, each canbe added by 0.1˜20 weight part per 100 weight part of the fraction ofthe oyster enzyme hydrate of the present invention or the peptideisolated therefrom.

The present invention also provides a method for treating cardiovasculardisease containing the step of administering the fraction of the oysterenzyme hydrate containing the said peptide comprising one of the aminoacid sequences represented by SEQ. ID. NO: 1˜NO: 12 to a subject havingcardiovascular disease.

The present invention also provides a method for treating cardiovasculardisease containing the step of administering at least one of the saidpeptides to a subject having cardiovascular disease.

The fraction of the oyster enzyme hydrate of the present invention orthe peptide isolated therefrom displays excellent ACE inhibitingactivity, suggesting that it could have efficient blood pressureregulating activity and antihypertensive effect thereby. Therefore, thefraction of the oyster enzyme hydrate and the peptide separated from thesame can be effectively used for the method for treating cardiovasculardisease.

The present invention also provide a method for preventingcardiovascular disease containing the step of administering the fractionof the oyster enzyme hydrate containing the said peptide comprising oneof the amino acid sequences represented by SEQ. ID. NO: 1˜NO: 12 to asubject.

The present invention also provides a method for preventingcardiovascular disease containing the step of administering at least oneof the said peptides.

The fraction of the oyster enzyme hydrate of the present invention orthe peptide isolated therefrom displays excellent ACE inhibitingactivity, suggesting that it could have efficient blood pressureregulating activity and antihypertensive effect thereby. Therefore, thefraction of the oyster enzyme hydrate and the peptide separated from thesame can be effectively used for the method for preventingcardiovascular disease.

The present invention also provides a use of the fraction of the oysterenzyme hydrate containing the said peptide comprising one of the aminoacid sequences represented by SEQ. ID. NO: 1˜NO: 12 as a pharmaceuticalcomposition for the prevention or treatment of cardiovascular disease.

The present invention also provides a use of at least one of the saidpeptides as a pharmaceutical composition for the prevention or treatmentof cardiovascular disease.

The present invention also provides a use of the fraction of the oysterenzyme hydrate containing the said peptide as a health food for theprevention or improvement of cardiovascular disease.

In addition, the present invention provides a use of at least one of thesaid peptides as a health food for the prevention or improvement ofcardiovascular disease.

The fraction of the oyster enzyme hydrate of the present invention orthe peptide isolated therefrom displays excellent ACE inhibitingactivity, suggesting that it could have efficient blood pressureregulating activity and antihypertensive effect thereby. Therefore, thefraction of the oyster enzyme hydrate and the peptide separated from thesame can be effectively used as an active ingredient of a pharmaceuticalcomposition for the prevention or treatment of cardiovascular disease ora health food for the prevention or improvement of cardiovasculardisease.

Practical and presently preferred embodiments of the present inventionare illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

Example 1 Preparation of oyster enzyme hydrate

To prepare the oyster enzyme hydrate of the present invention, oysterprotein was hydrolyzed by using an enzyme.

Particularly, 3 kg of the cultured oyster (purchased from the OysterHanging Fisheries Cooperatives, Tongyeong-si, Gyeongsangnam-do, Korea)was parboiled for 3 minutes and then water was drained by a strainer.Water was added to the oyster twice the volume of the oyster, followedby grinding with a grinder (M-12S, Hankook Fujee Machinery Co., Ltd.,Korea) at 3000 rpm for 2 minutes. The grinded oyster andtransglutaminase (TGase; Ajinomoto Co., Inc., Japan) were loaded in 5 LJar fermenter (Korea Fermenter, Korea), followed by reaction at 30° C.for 1 hour with stirring at 150 rpm. The reaction mixture wasinactivated by heating at 100° C. for 1 hour. Protamex(Nobozymes-Korea), the Bacillus originated protease was added thereto,followed by reaction at 40° C. for 1 hour. As a result, the primaryoyster enzyme hydrate was obtained. The primary oyster enzyme hydratewas treated at 100° C. for 1 hour to make Protamex inactivated. Then,Neutrase (Nobozymes-Korea) was added thereto, followed by reaction at50° C. for 1 hour. As a result, the secondary oyster enzyme hydrate wasobtained. The secondary oyster enzyme hydrate was treated at 100° C. for1 hour to make Neutrase inactivated. Then, the secondary oyster enzymehydrate was centrifuged to obtain supernatant. Ethanol was added to thesupernatant at the volume of 60% (v/v) at 4° C., followed by reactionfor 1 hour to induce the precipitation of ethanol-insoluble materialsand the remaining proteins. Centrifugation was performed at 8000×g for25 minutes to obtain supernatant. The obtained supernatant was filteredwith 0.45 μm membrane filter and ethanol was evaporated by using arotary vacuum evaporator (N-1 type, EYELA, Japan) at 40° C. Theresultant oyster enzyme hydrate was freeze-dried or spray-dried andstored.

Example 2 Purification of Functional Peptide Having Angiotensin-IConverting Enzyme (ACE) Inhibiting Activity <2-1> Purification by AnionExchange Chromatography

To purify the functional peptide having ACE inhibiting activity from theoyster enzyme hydrate, anion exchange chromatography was performed.

Particularly, 0.5 g of the oyster enzyme hydrate prepared in Example<1-1> was dissolved in 12 ml of 20 mM Tris-HCl buffer (pH 7.5), whichwas loaded in Q-Sepharose column (16×100 mm; GE Healthcare-Korea),followed by anion exchange chromatography under the conditions shown inTable 1. Then the oyster enzyme hydrate fractions were obtained, 2 mleach, and the detected peptides were confirmed.

As a result, as shown in FIG. 1, the fractions containing the peptideseparated from the oyster enzyme hydrate were obtained (FIG. 1).

TABLE 1 Chromatography condition for functional peptide purificationFlow Detection rate wavelength Chromatography Column Solvent (ml/min)(nm) Anion Q- A*: 20 mM 1 254 exchange Sepharose Tris-HCl chromatographycolumn buffer (16 × 100 (pH 7.5) mm) B: containing 0.75M NaCl 20 mMTris- HCl buffer (pH 7.5) Size Superdex 20 mM Tris- 0.5 216 andexclusion peptide HCl buffer 254 chromatography ^(a) (10 × 300 (pH 7.5)mm) Reverse- Source A**: 0.1% 1 216 and phase 5RPC ST TFA^(c) 254chromatography ^(b) (4.6 × 150 solution mm) B: 0.09% TFA and 60% ACN^(d)solution ^(a) size exclusion chromatography ^(b) reverse-phasechromatography ^(c)TFA: trifluoroacetic acid ^(d)ACN: acetonitrile*solvent density gradient: 100 ml/100 min., 0~100% solvent B densitygradient. **solvent density gradient: 90 ml/90 min., linear gradation.

<2-2> Confirmation of ACE Inhibiting Activity

To investigate ACE inhibiting activity of the fraction obtained inExample <2-1>, ACE activity was measured by using the method of Wu et al(Wu et al., 2002; J Chromatography A 950:125-130) with slightmodifications.

Particularly, 0.1 M borate buffer (pH 8.3) containing 0.3 M NaCl, 5 mMN-benzoyl-Gly-His-Leu (HHL; Sigma; product #H1635), and 0.25 mUnit ACE(Sigma, USA) was prepared as the reaction solution. Then, 40 μl of thefraction prepared in Example <2-1> was mixed with 150 μl of the reactionsolution above, followed by reaction with stirring in a 37° C. waterbath for 30 minutes. Upon completion of the reaction, 150 μl of 1 M HClwas added to terminate ACE reaction, followed by centrifugation at10,000 rpm for 10 minutes to obtain supernatant using a centrifuge(product name: 5415C; Eppendorf, Hamburg, Germany). 20 μl of theobtained supernatant was loaded on high performance liquidchromatography (HPLC) equipped with reverse-phase column (Watchers 120ODS-AP, 4.6×250 mm, 5 μm; Daiso, Japan), from which the content ofhippuric acid (HA) dissociated from HHL by ACE was measured. For HPLC,0.1% TFA aqueous solution was used as solvent A and acetonitrilecontaining 0.1% TFA was used as solvent B for the linear gradation underthe conditions of 5˜60% solvent B/20 minutes. During the elution at 1ml/min, OD₂₂₈ was measured. Then, ACE inhibiting activity was calculatedby the below mathematical formula 1. Dose-dependent ACE inhibitingactivity was analyzed by linear regression analysis (JMP statisticspackage ver. 7, SAS Institute, Cary, N.C., USA) and then IC₅₀, theconcentration of the sample that was able to inhibit ACE activity by50%, was calculated to investigate ACE inhibiting activity. For thenegative control, 20 μl of 0.1% TFA aqueous solution was used instead ofthe sample. Then, ACE inhibiting activity was measured by the samemanner as described above.

ACE inhibiting activity (%)=HAo−HA/HAo×100  [Mathematical Formula 1]

HAo: HA conc. of negative control

HA: HA conc. of sample

As a result, as shown in Table 2, ACE inhibiting activity was observedin the samples of fractions 2, 3, and 4.

<2-3> Purification by Size Exclusion Chromatography

To purify the functional peptide having ACE inhibiting activity from theoyster enzyme hydrate, size exclusion chromatography was performed.

Particularly, the fractions 2, 3, and 4 selected in Example <2-2> wereconcentrated in Speed Vacuum Concentrator (scan speed 40, Labgene Aps,Denmark). 200 μl of the concentrated sample was loaded on superdexpeptide column (10×300 mm; GE Healthcare-Korea), followed by sizeexclusion chromatography over the molecular weight under the conditionsshown in Table 1. The fraction of each sample was investigated by thesame manner as described in Example <2-2> to confirm ACE inhibitingactivity.

As a result, as shown in FIG. 1 and Table 2, the fractions 2-1, 2-2,2-3, 3-1, 3-2, 4-1, and 4-2 containing the said peptide were selectedthrough size exclusion chromatography (FIG. 1). These fractions wereconfirmed to have ACE inhibiting activity (Table 2).

<2-4> Purification by Reverse-Phase Chromatography

To purify the functional peptide having ACE inhibiting activity from theoyster enzyme hydrate, reverse-phase HPLC was performed.

Particularly, the fractions 2-1, 2-2, 2-3, 3-1, 3-2, 4-1, and 4-2selected in Example <2-3> were loaded in source 5 RPC ST column (4.6×150mm; GE Healthcare-Korea), followed by reverse-phase chromatography overthe molecular weight under the conditions shown in Table 1. The fractionof each sample was investigated by the same manner as described inExample <2-2> to confirm ACE inhibiting activity.

As a result, as shown in FIG. 1 and Table 2, the fractions containingthe said peptide were selected through reverse-phase chromatography(FIG. 1). These fractions were confirmed to have ACE inhibiting activity(Table 2).

TABLE 2 Fraction name, fraction number, ACE inhibiting activity andpeptide sequence according to purification stage ACE inhibiting FractionFraction activity Purification name number (%) Peptide Anion exchange 225, 26, 27 4.0 chromatography 3 29 4.9 4 30, 31 6.7 Size exclusion 2-119, 20 19.0 chromatography 2-2 21 17.0 2-3 23 15.0 3-1 20 15.7 3-2 2117.8 4-1 20, 18.3 4-2 21, 22 18.6 Reverse-phase 2-1-3 20, 21 34.0 AFN,FYN chromatography 2-2-2 21, 21 27.7 TAY 2-3-2 16, 17 30.4 KY 3-1-4 20,21 54.5 AFY 3-2-2 14 44.3 VK 4-1-1 18, 19 34.4 PGN, GPN 4-2-1 16 26.5MC, PH, SF, YA

Example 3 Investigation of Mass and Sequence of the Functional PeptideHaving ACE Inhibiting Activity

To screen the functional peptide having ACE inhibiting activityseparated from the oyster enzyme hydrate, the mass and the amino acidsequence of the said peptide were investigated by Edman degradation andMALDI/TOF (Matrix-Assisted Laser Desorption Ionization/Time-Of-FlightMass Spectroscopy).

Particularly, 7 samples displaying ACE inhibiting activity selected inExample <2-4> were completely dried in a vacuum centrifugalconcentrator, to which 20 μl of 0.1% TFA aqueous solution was addeduntil the samples were fully dissolved. Then, 50% acetonitrile was addedto induce activation and the dissolved samples were loaded in ZipTip C18column (Fierce, product #: 87782) equilibrated with 0.1% TFA aqueoussolution. Then, the column was washed with 0.1% TFA aqueous solution 2˜3times. The peptide was eluted by using 70% acetonitrile containing 0.1%TFA to eliminate the remaining salts. 10 μl of the eluted peptidesolution was loaded on biobrene (AB Systems, USA) pre-treatedmicro-filter, and the filter was dried over argon gas. When the filterwas dried, it was loaded in the cartridge and the amino acid sequencethat composed the functional peptide was analyzed by pulsed-liquidmethod using ABI482 automated protein sequencer (Applied Biosystems,USA).

The 7 samples displaying ACE inhibiting activity selected in Example<2-4> proceeded to the mass spectrometer Q-TOF2 (Micromass, UnitedKingdom) based on electrospray Ionization (ESI) to perform datadependent MS/MS via Nano-ESI-interface.

As a result, as shown in FIG. 2 and Table 3, total peptides composed ofthe amino acid sequences represented by SEQ. ID. NO: 1˜NO: 12 wereconfirmed (Table 3 and FIG. 2).

TABLE 3 Amino acid sequences of the functional peptides showing ACEinhibiting activity Peptide Sample Amino acid sequence TAY (SEQ. ID.1-2-2 threonine-alanine-tyrosine NO: 1) VK (SEQ. ID. 2-2-2 valine-lysineNO: 2) KY (SEQ. ID. 1-3-2 lysine-tyrosine NO: 3) YA (SEQ. ID. 3-2-1tyrosine-alanine NO: 4) FYN (SEQ. ID. 1-1-3 phenylalanine-tyrosine- NO:5) asparagine AFY (SEQ. ID. 2-1-4 alanine-phenylalanine-tyrosine NO: 6)MC (SEQ. ID. 3-2-1 methionine-cysteine NO: 7) GPN (SEQ. ID. 3-1-1glycine-proline-asparagine NO: 8) AFN (SEQ. ID. 1-1-3alanine-phenylalanine- NO: 9) asparagine PGN (SEQ. ID. 3-1-1proline-glycine-asparagine NO: 10) PH (SEQ. ID. 3-2-1 proline-histidineNO: 11) SF (SEQ. ID. 3-2-1 serine-phenylalanine NO: 12)

Example 4 Synthesis of the Functional Peptide Showing ACE InhibitingActivity and Confirmation of the Activity <4-1> Synthesis of theFunctional Peptide

To investigate whether or not the functional peptide separated from theoyster enzyme hydrate could have ACE inhibiting activity, Fmoc-SPPS(Fluorenylmethyloxycarbonyl chloride-Solid phase peptide synthesis) wasperformed to prepare synthetic peptides.

Particularly, the amino acid in which C-terminal was binding to resin,N-terminal was protected by Fmoc, and also the residue was protected bysuch protection group as trityl (Trt), t-butyloxycarbonyl (Boc), ort-butyl (t-Bu) was prepared as the material for the amino acid synthesisthat composed the peptide sequence confirmed in Example 3. The preparedamino acid was added to coupling reagent containing2-(1H-Benzotirazloe-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), hydroxybenzotriazole (HOBt) and N-methylmorpholine (NMM),followed by reaction using an automatic synthesizer (ASP48S; Peptron,Korea) at room temperature for 2 hours. Dimethylformamide (DMF)containing 20% piperidine was added thereto, followed by reaction atroom temperature for 5 minutes, during which Fmoc was eliminated,resulting in the synthesis of the peptide. The peptide of the presentinvention was synthesized by repeating the process. Next, TFA,1,2-ethanedithiol (EDT), Thioanisole, Triisopropylsilane (TIS), andwater were mixed at the ratio of 90%, 2.5%, 2.5%, 2.5%, and 2.5% (v/v),to which the synthesized peptide was added. The resin conjugated atC-terminal and the protection group in the residue were eliminated.Reverse-phase HPLC was performed using Vydac Everest C18 column (22×250mm, 10 μm; Grace, USA) under the linear gradation condition with 40%acetonitrile solution containing 0.1% TFA, resulting in the separationand purification of the peptide. The purified peptide was confirmed byLC/MS using Agilent HP1100 series (Agilent, USA).

As a result, 12 synthetic peptides composed of the same amino acidsequences as those composing the functional peptides separated from theoyster enzyme hydrate were prepared.

<4-2> Confirmation of ACE Inhibiting Activity and Cytotoxicity of theSynthetic Peptide

To investigate if the functional peptide separated from the oysterenzyme hydrate could have ACE inhibiting activity, the synthetic peptidewas tested and MTT assay was performed to confirm the cytotoxicity.

Particularly, ACE inhibiting activity of the peptide synthesized inExample <4-1> was investigated by the same manner as described inExample <2-2>. To measure the cytotoxicity, the liver cancer cell lineHepG2 was inoculated in MEM supplemented with antibiotics and 10% fetalbovine serum (FBS), followed by culture at 37° C. with 5% CO₂. When thecells were grown to 80% confluency, the cells were washed withphosphate-buffered saline-ethylenediaminetetraacetic acid (PBS-EDTA) andthen treated with trypsin, followed by sub-culture. The medium wasreplaced every 48 hours during the cell culture. The cells weredistributed in a 96-well plate at the density of 1×10⁵ cells/id, 100μl/well, followed by culture for 24 hours to attach the cells onto theplate. Then, the medium was discarded. The peptide synthesized inExample <4-1> was dissolved in MEM containing 0.2% PBS at theconcentrations of 10 μg/ml, 50 μg/ml, 100 μg/ml, or 200 μg/ml. Thepeptide was treated to the cells above, followed by further culture for24 hours. Upon completion of the culture, the cells were washed with PBStwice, and then treated with MTT reagent. The cells were furthercultured for 2 hours and OD₄₉₀ was measured with ELISA plate reader.

As a result, as shown in Table 4, it was confirmed that all of those 12functional peptides did not show cytotoxicity but had ACE inhibitingactivity (Table 4).

TABLE 4 ACE inhibiting activity and cytotoxicity of functional peptideACE inhibiting activity Peptide IC₅₀ (μM) IC₅₀ (μg/μl) Cytotoxicity (%)TAY 16.7 2.18 101.2 ± 6.1 VK 29.0 2.63 103.2 ± 3.9 KY 51.5 5.90 105.4 ±5.7 YA 93.9 8.76 103.8 ± 7.7 FYN 68.2 11.17 109.5 ± 3.0 AFY 75.6 11.18106.7 ± 7.4 MC 153.8 14.36 100.6 ± 4.6 GPN 208.5 22.09 108.3 ± 1.0 AFN222.9 28.89 108.1 ± 4.4 PGN 371.9 39.39 103.9 ± 6.2 PH 537.1 50.13 113.7± 6.0 SF 646.3 60.33  99.0 ± 1.2

Example 5 In Vivo Blood Pressure Regulation by the Oyster Enzyme Hydrateand the Functional Peptide Separated Therefrom <5-1> Separation of theOyster Enzyme Hydrate According to the Molecular Weight

To investigate in vivo blood pressure regulation effect of the oysterenzyme hydrate according to the molecular weight, the oyster enzymehydrate was divided into two groups; one with the molecular weight of atleast 10 kD and the other with the molecular weight of under 10 kD.

Particularly, the oyster enzyme hydrate prepared in Example 1 wasfiltered by ultrafiltration (Labscale TFF system, Millipore, USA) usingBiomax 10 ultrafiltration membrane (Millipore, USA) and pellicon XLultrafiltration membrane (Millipore, USA).

As a result, the oyster enzyme hydrate having either 10 kD or highermolecular weight or 10 kD or lower molecular weight was obtained fromthe whole oyster enzyme hydrate.

<5-2> Investigation of In Vivo Blood Pressure Regulation Effect by aSingle Administration

To investigate in vivo blood pressure regulation effect of thefunctional peptide separated from the oyster enzyme hydrate by a singleadministration, the peptide was administered to the hypertension animalmodel once (single administration), followed by the investigation ofblood pressure.

Particularly, male spontaneously hypertensive rats (SHR; Central Lab.Animal Inc.) at 11 weeks and Wistar rats (Central Lab. Animal Inc.) werepurchased, which were raised in a laboratory animal room in which thetemperature was regulated at 22±3° C., the humidity was controlled at50±5%, and the light/dark cycle was set at h/12 h. Feeds and drinkingwater were provided freely. After a week of adaptation, the animals weredivided into the experimental and the control groups and used for theexperiment according to the guide line of Association for Assessment andAccreditation of Laboratory Care International approved by InstitutionalAnimal Care and Use Committee, Kyunghee University College of Pharmacy.The experimental and the control groups were divided by the conditionsshown in Table 5. The animals were orally or intraperitoneallyadministered with the oyster enzyme hydrate prepared in Example 1, theoyster enzyme hydrate having the molecular weight of at least 10 kD orup to 10 kD prepared in Example <5-1>, the peptide prepared in Example<4-1>, or Captopril (Boryung Co., Ltd., Korea). Then the animals werefixed for 20 minutes in a rat temperature control unit in which thetemperature was maintained at 42° C. at the time point of 0, 3, 6, 9,12, and 24 h, to let the tail vessel fully expanded. Then, systolicpressure was measured to investigate the antihypertensive activity.

TABLE 5 Conditions for the experimental and the control groups toconfirm the in vivo blood pressure regulation effect by a singleadministration Administration Animal dose^(a) Wistar Normal Saline 2 mlRat control SHR Negative Saline 2 ml control Positive Captopril 8 mg/kgcontrol Experimental Oyster Whole 100 mg/kg group enzyme Over hydrate 10kD Less 10 kD Functional TAY 50 μg/kg peptide VK KY YA FYN AFY MC^(a)The dose of the sample presented as mg/kg for the positive controland the experimental groups was mixed with 2 ml of saline for theadministration.

As a result, as shown in FIG. 3 and FIG. 4, in the positive controltreated with Captopril, the conventional antihypertensive agent, theblood pressure lowering effect was only 18˜19% by that of the negativecontrol hypertensive rat model. In the meantime, when the oyster enzymehydrate was administered, the blood pressure lowering effect wasincreased to 33˜38% by the negative control. In particular, when theoyster enzyme hydrate having the molecular weight of up to 10 kDa, bloodpressure of the experimental group was significantly lowered almost backto that of the normal control until 12 hours from the administration ofthe oyster enzyme hydrate (FIG. 3).

In the experimental group treated with the functional peptide of theinvention, a significant blood pressure lowering effect was observed,compared with that of the negative control hypertensive model. Inparticular, when peptide YA was administered, the blood pressurelowering effect after 6 hours from the administration was 33.9%, whichwas the highest effect of all (FIG. 4).

<5-3> Investigation of In Vivo Blood Pressure Regulation Effect by theRepeated Administration

To investigate in vivo blood pressure regulation effect of thefunctional peptide separated from the oyster enzyme hydrate by therepeated administration, the peptide was administered to thehypertension animal model repeatedly, followed by the investigation ofblood pressure.

Particularly, the same rats as described in Example <5-2> were purchasedand raised under the same condition. The rats were divided into theexperimental and the control groups according to the conditions as shownin Table 6, to which the oyster enzyme hydrate having the molecularweight of up to 10 kD prepared in Example <5-1>, the peptide YA preparedin Example <4-1>, or Val-Tyr, the antihypertensive material separatedfrom sardine hydrolysate were administered orally, every morning at 10am, for 4 weeks. Systolic pressure was measured by the same manner asdescribed in Example <5-2> on week 0, week 1, week 2, week 3, and week4. 4 weeks after the administration began, the rats were sacrificed andblood was extracted from the inferior vena cava. The blood sample wascentrifuged at 3,000 rpm for 10 minutes, and as a result serum wasobtained and stored at −80° C. The levels of blood ACE andangiotensin-II in the stored serum were measured by using a commercialELISA kit (USCN Life Science, China) according to the manufacturer'sprotocol.

TABLE 6 Conditions for the experimental and the control groups toconfirm the in vivo blood pressure regulation effect by the repeatedadministration Administration Animal dose^(a) Wistar Normal Saline 2 mlRat control SHR Negative Saline 2 ml control Positive Val-Tyr 50 μg/kgcontrol Experimental Oyster Whole 100 mg/kg group enzyme Less hydratethan 10 kD Functional YA 50 μg/kg peptide ^(a)The dose of the samplepresented as mg/kg for the positive control and the experimental groupswas mixed with 2 ml of saline for the administration.

As a result, as shown in FIGS. 5˜7, a significant blood pressurelowering effect was observed in all the experimental groups, comparedwith the negative control hypertensive rat model. Particularly, in thegroups treated with peptide YA and the whole oyster enzyme hydrate,blood pressure did not increase for 2 weeks from the administration. Inthe group treated with the oyster enzyme hydrate of up to 10 kD, bloodpressure was slightly increased for 2 weeks from the administration, butfrom the third week the blood pressure lowering effect began to display,which was significantly increased further on (FIG. 5).

The level of blood ACE was reduced in all the experimental groups,compared with that of the negative control hypertensive group (FIG. 6).The level of blood angiotensin-II was also significantly reduced in allthe experimental groups, compared with that of the negative controlgroup, which was more significant than that of the positive controladministered with Val-Tyr, the antihypertensive functional peptideseparated from sardine hydrolysate. Particularly, in the group treatedwith the oyster enzyme hydrate of up to 10 kD, the level of bloodangiotensin-II was almost same as that of the normal control group (FIG.7).

Example 6 Hypertension Preventive Effect of the Oyster Enzyme Hydrateand the Functional Peptide Separated from the Same

To investigate the hypertension preventive effect of the functionalpeptide separated from the oyster enzyme hydrate, the peptide wasadministered to the hypertension animal model, and then theantihypertensive effect was observed.

Particularly, male Wistar rats at 12˜16 weeks were purchased and raisedin a laboratory animal room in which the temperature was regulated at22±3° C., the humidity was controlled at 50±5%, and the light/dark cyclewas set at 12 h/12 h. Feeds and drinking water were provided freely.After a week of adaptation, the rats were divided into the experimentaland the control groups (8 rats/group) according to the conditions listedin Table 7. To induce hypertension, the rats of the experimental andcontrol groups were administered with nitro-L-arginine methyl ester(L-NAMA; Fluka) at the concentration of 40 mg/kg via intravenousinjection every day. Then, the rats were orally administered with theoyster enzyme hydrate prepared in Example 1, the oyster enzyme hydrateof up to 10 kD prepared in Example <5-1>, the peptide YA prepared inExample <4-2>, Captopril, or Val-Tyr. Systolic pressure was measured bythe same manner as described in Example <5-2> on week 0, week 1, week 2,week 3, and week 4. Then average systolic pressure of each control andexperimental group was calculated.

TABLE 7 Conditions for the experimental and the control groups toconfirm the in vivo hypertension preventive effect of the oyster enzymehydrate and the functional peptide separated therefrom AdministrationAnimal dose^(a) Wistar Normal Saline 2 ml Rat control Negative Saline 2ml control Positive Captopril 8 mg/kg control Sardine peptide 100 mg/kgVal-Tyr 50 μg/kg Experimental Oyster Whole 100 mg/kg group enzyme Less100 mg/kg hydrate than 10 kD Functional YA 50 μg/kg peptide ^(a)The doseof the sample presented as mg/kg for the positive control and theexperimental groups was mixed with 2 ml of saline for theadministration.

As a result, as shown in FIG. 8, normal rats were induced withhypertension by treating L-NAMA. Compared with the positive controlgroup, the blood pressure increase was significantly suppressed in therat group administered with the oyster enzyme hydrate and the functionalpeptide (FIG. 8).

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended Claims.

What is claimed is:
 1. A method for treating cardiovascular diseasecontaining the step of administering at least one of the peptides havingthe amino acid sequences represented by SEQ ID NOs: 1˜12 to a subjecthaving cardiovascular disease.
 2. The method according to claim 1,wherein the peptide is separated from the oyster enzyme hydrate.
 3. Themethod according to claim 2, wherein the enzyme is protease.
 4. Themethod according to claim 1, wherein the peptide inhibiting angiotensinconverting enzyme (ACE).
 5. The method according to claim 1, wherein thecardiovascular disease is one or more diseases selected from the groupconsisting of hypertension, heart disease, stroke, thrombosis, anginapectoris, heart failure, myocardial infarction, atherosclerosis, andarteriosclerosis.
 6. A method for treating cardiovascular diseasecontaining the step of administering a fraction of the oyster enzymehydrate containing the peptide comprising one of the amino acidsequences represented by SEQ ID NOs: 1˜NO: 12 to a subject havingcardiovascular disease.
 7. The method according to claim 6, wherein thefraction is up to 10 kD.
 8. The method according to claim 6, wherein thecardiovascular disease is one or more diseases selected from the groupconsisting of hypertension, heart disease, stroke, thrombosis, anginapectoris, heart failure, myocardial infarction, atherosclerosis, andarteriosclerosis.
 9. A method for inhibiting angiotensin convertingenzyme (ACE) containing the step of administering at least one of thepeptides having the amino acid sequences represented by SEQ ID NOs: 1˜12to a subject.